Highly Selective Anti-Cancer Agents Targeting Non-Small Cell Lung Cancer and Other Forms of Cancer

ABSTRACT

Described herein are analogues of 2-methyl-3-(2-ethynylthiazol-4-yl)cyclopent-2-enol and the corresponding ketone 3-(2-ethynylthiazol-4-yl)-2-methylcyclopent-2-enone, the analogues having terminal alkyne groups at the 2-position of the thiazole ring. These drug-like molecules, referred to as CETZOLE compounds, are useful to treat non-small cell lung cancer and other forms of cancer. Methods of making and using the compounds, methods of treating various diseases, pharmaceutical compositions, and kits are also disclosed.

RELATED APPLICATIONS

The present application claims priority to U.S. Provisional ApplicationSer. No. 61/755,808, filed under 35 U.S.C. §111(b) on Jan. 23, 2013, theentire disclosure of which is incorporated herein by reference.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

The invention was not made with any government support. The governmenthas no rights in the invention.

BACKGROUND OF THE INVENTION

Lung cancer is the leading cause of cancer death in the United States.The American Cancer Society estimates more than 240,000 new cases oflung cancer will occur, and more than 140,000 deaths will be attributedto lung cancer, in 2012. Lung cancer is categorized as either non-smallcell lung carcinoma (NSCLC) or small cell lung carcinoma, with NSCLCrepresenting more than 80% of cases.

Current treatments for lung cancer include surgery, radiation, classicalchemotherapeutic agents (platinum compounds, taxanes), and targetedtherapies (inhibitors of VEGFR, EGFR, IGFR, HDACS, and the proteasome).However, despite advances in treatment, five-year survival rates areabout 16%. Numerous clinical trials evaluating classical chemotherapydrugs for lung cancer indicate that a therapeutic plateau with currentdrugs may have been reached. Therefore, there is a need for new drugsfor the treatment of lung cancer that have different mechanisms ofaction.

SUMMARY OF THE INVENTION

Provided herein is a compound having the structural formula of Formula Ior Formula II:

wherein n=1 or 2; X is N or CH; Y is S, O, or NH; R is ═O; 2H; —OR₃;wherein R₃ is hydrogen, alkyl, aryl, glycosyl, or polyether groups;(CO)OR₄, wherein R₄ is hydrogen, alkyl, aryl, or aralkyl groups; —NR₅R₆,wherein R₅ and R₆ are H, alkyl, aryl, or aralkyl groups; or ═N—OR₇,wherein R₇ is hydrogen, alkyl, aryl, aralkyl, glycosyl, or polyethergroups; R₁ is hydrogen, alkyl-, aryl-, or aralkyl; Z is absent orselected from the group consisting of: halide, wherein R₂ is thenabsent; oxygen; nitrogen, wherein there are then two R₂ groups; (CO)O—;O(CO)—; O(CO)O—; (CO)N<, wherein there are then two R₂ groups; NH(CO)—;and NH(CO)N<, wherein there are then two R₂ groups; wherein when Z isabsent, R₂ connects directly to a ring; and R₂ is hydrogen, alkyl-,aryl-, aralkyl-, glycosyl-, or polyether-, wherein multiple occurrencesof R₂ are the same or different; and salts, stereoisomers, racemates,prodrugs, solvates, and hydrates thereof.

In certain embodiments, the compound has the structural formula ofFormula III:

and salts, stereoisomers, racemates, prodrugs, solvates, and hydratesthereof.

In certain embodiments, the compound has the structural formula ofFormula IV:

wherein R is H,C₆H₅CH₂, or polyether; and salts, stereoisomers,racemates, prodrugs, solvates, and hydrates thereof.

In certain embodiments, the compound further comprises a protectinggroup. In particular embodiments, the protecting group is selected fromthe group consisting of: benzyl, t-butyl dimethyl silyl, isobutyryl,acetyl, phenoxyacetyl, allyloxycarbonyl (AOC), diisobutylformamidine,benzoyl, formyl, trifluoroacetyl, benzyloxycarbonyl (Cbz), substitutedbenzyloxycarbonyl, dimethoxy trityl (DMT), monomethoxytrityl (MMT), and9-fluorenylmethyloxycarbonyl (Fmoc), t-butyloxycarbonyl (Boc), isopropyloxycarbonyl, cyclohexyloxycarbonyl, and a combination thereof. Incertain embodiments, the compound comprises at least one hydroxylprotecting group. In particular embodiments, the at least one hydroxylprotecting group is selected from the group consisting of alkyl silylgroups, alkyl ethers, and esters.

Further provided is a compound having a4-cyclopentyenyl-2-ethynylthiazole skeleton and a terminal alkyne at the2-position of the thiazole ring.

Further provided herein is a method of making a CETZOLE compoundcomprising subjecting a dibromothiazole to Sonogashira coupling withTMS-acetylene to obtain a silylated bromothiazole alkyne, subjecting thesilylated bromothiazole alkyne to Stille coupling with an iodoalkene toobtain a silylated CETZOLE compound, and desilylating the silylatedCETZOLE compound to obtain a CETZOLE compound. In certain embodiments,the dibromothiazole consists essentially of 2,4-dibromothiazole. Incertain embodiments, the silylated bromothiazole alkyne consistsessentially of 2-((trimethylsilyl)ethynyl)-4-bromothiazole. In certainembodiments, the iodoalkene consists essentially of3-iodo-2-methylcyclopent-2-enone. In certain embodiments, thedesilylating comprises mixing a solution of K₂CO₃ and the silylatedCETZOLE compound in methanol for a period of time.

In certain embodiments, the method further comprises the step ofsubjecting the CETZOLE compound to stereoselective CBS reduction toobtain a second CETZOLE compound. In certain embodiments, thestereoselective CBS reduction comprises adding borane-Me₂S to a solutionof R-2-methyl-CBS-oxazaborolidine and the CETZOLE compound.

In certain embodiments, the method further comprises the steps oftreating the CETZOLE compound with lithium diisopropylamide and benzylbromide to obtain a silylated benzyl CETZOLE derivative, anddesilylating the silylated benzyl CETZOLE derivative to obtain abenzylated CETZOLE compound. In certain embodiments, the silylatedbenzyl CETZOLE derivative consists essentially of5-benzyl-2-methyl-3-(2-((trimethylsilyl)ethynyl)thiazol-4-yl)cyclopent-2-enone.In certain embodiments, the desilylating comprises mixing K₂CO₃ and MeOHwith the silylated benzyl CETZOLE derivative. In certain embodiments,the benzylated CETZOLE compound consists essentially of5-benzyl-3-(2-ethynylthiazol-4-yl)-2-methylcyclopent-2-enone.

Further provided herein is a pharmaceutical composition comprising aCETZOLE compound as described herein and a pharmaceutically acceptablecarrier, diluent, or excipient.

Further provided herein is a method of treating a mammalian disease, themethod comprising administering a therapeutically effective amount of apharmaceutical composition disclosed herein to a subject in needthereof. In certain embodiments, the disease comprises cancer. Incertain embodiments, the disease is non-small cell lung carcinoma(NSCLC). In certain embodiments, the disease is renal cancer. In certainembodiments, the disease is ovarian cancer. In certain embodiments, thedisease is CNS cancer. In certain embodiments, the disease is melanoma.

Further provided herein is a method of treating cancer, the methodcomprising administering to a subject an agent that is lethal to cellsby a mechanism that does not depend on apoptosis, wherein the agentcomprises a compound having a 4-cyclopentyenyl-2ethynylthiazole skeletonand a terminal alkyne at the 2-position of the thiazole ring.

Further provided herein is a kit for preparing a CETZOLE compound, thekit comprising a first container housing a dibromothiazole, and a secondcontainer housing TMS-acetylene. In certain embodiments, the kit furthercomprises one or more of an iodoalkene, lithium diisopropylamide, orbenzyl bromide.

Further provided is a composition resulting by ingestion in a compoundas described herein.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file may contain one or more drawings executedin color and/or one or more photographs. Copies of this patent or patentapplication publication with color drawing(s) and/or photograph(s) willbe provided by the U.S. Patent and Trademark Office upon request andpayment of the necessary fees.

FIG. 1: Scheme of the synthetic route for compounds 2 and 3.

FIG. 2: The effect of compounds 2, 3, and 4 on cell viability of variouscell lines.

FIGS. 3A-3C: The effect of compound 2 (10 μM) on tubulin andmitochondrial function. FIG. 3A shows NCI-H522 cells exposed to compound2 for 8 hours and stained with antibodies to tubulin. FIG. 3B showsNCI-H522 cells treated 4 times with 2 and stained with TMRE to indicatemitochondrial potential. Arrowheads: two cells that have lostmitochondrial membrane potential; phase contrast indicates these aredead cells. FIG. 3C shows quantitation of TMRE staining from microscopicimages.

FIG. 4: The effects of compounds 1f, 1c, and 1a on cell viability, incomparison with the effects on cell viability of compound 2 and thedrugs hydroxyurea, taxol®, and adriamycin.

FIGS. 5A-5C: The effect of compound 1f on NCI-H522 cells at 10 μM. FIG.5A shows cells plated in a slide-flask and analyzed by time-lapsemicroscopy. Interval between images shown is 1 hour. FIG. 5B shows aKaplan-Meier plot of NCI-H522 cells exposed to compound 1f. FIG. 5Cshows dose response of HOP62 and NCI-H522 exposed to compound 1f.

FIG. 6: Scheme showing the synthesis of5-benzyl-3-(2-ethynylthiazol-4-yl)-2-methylcyclopent-2-enone 4.

DETAILED DESCRIPTION OF THE INVENTION

For convenience, various terms used herein are defined prior to furtherdescription of the various embodiments of the present disclosure.

Unless stereochemistry is specifically indicated, all stereoisomers ofthe compounds herein are included, as pure compounds as well as mixturesthereof.

The term “composition” as used herein is intended to encompass a productcomprising the specified ingredients in the specified amounts, as wellas any product that results, directly or indirectly, from combinationsof the specified ingredients in the specified amounts.

The term “protecting group” as used herein refers to a group which isintroduced onto a functional group in a compound and which modifies thatfunctional group's chemical reactivity. Typically, the protecting groupmodifies the functional group's chemical activity in such a way that itrenders the functional group chemically inert to the reaction conditionsused when a subsequent chemical transformation is effected on thecompound. A “hydroxyl protecting group” is accordingly a protectinggroup which is introduced onto a hydroxyl group in a compound.

The term “alkyl” refers to monovalent alkyl groups having from 1 to 50carbon atoms, preferably having from 1 to 10 carbon atoms, and morepreferably having from 1 to 6 carbon atoms. This term is exemplified bygroups such as methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl,n-hexyl, and the like. “Substituted alkyl” refers to an alkyl group,preferably of from 1 to 10 carbon atoms, having from 1 to 3 substituentsselected from the group consisting of alkoxy, substituted alkoxy, acyl,acylamino, amino, aminoacyl, aminocarboxy esters, cyano, cycloalkyl,halogen, hydroxyl, carboxyl, carboxylalkyl, oxyacyl, oxyacylamino,thiol, thioalkoxy, substituted thioalkoxy, aryl, heteroaryl,heterocyclic, aryloxy, thioaryloxy, heteroaryloxy, thioheteroaryloxy,nitro, and mono- and di-alkylamino, mono- and di-(substitutedalkyl)amino, mono- and di-arylamino, mono- and di-heteroarylamino, mono-and di-heterocyclic amino, and unsymmetric di-substituted amines havingdifferent substituents selected from alkyl, substituted alkyl, aryl,heteroaryl, and heterocyclic.

The term “aryl” refers to an unsaturated aromatic carbocyclic group,preferably of from 6 to 14 carbon atoms, having a single ring (e.g.,phenyl) or multiple condensed rings (e.g., naphthyl or anthryl),preferably having from 1 to 3 rings. Preferred aryls include phenyl,naphthyl, and the like. Unless otherwise constrained by the definitionfor the aryl substituent, such aryl groups can optionally be substitutedwith from 1 to 3 substituents selected from the group consisting ofhydroxy, acyl, alkyl, alkoxy, alkenyl, alkynyl, substituted alkyl,substituted alkoxy, substituted alkenyl, substituted alkynyl, amino,aminoacyl, aminocarboxy esters, alkaryl, aryl, aryloxy, carboxyl,carboxylalkyl, acylamino, cyano, halo, nitro, heteroaryl, heterocyclic,oxyacyl, oxyacylamino, thioalkoxy, substituted thioalkoxy,trihalomethyl, mono- and di-alkylamino, mono- and di-(substitutedalkyl)amino, mono- and di-arylamino, mono- and di-heteroarylamino, mono-and di-heterocyclic amino, and unsymmetric di-substituted amines havingdifferent substituents selected from alkyl, substituted alkyl, aryl,heteroaryl, heterocyclic, and the like. Preferred substituents includealkyl, alkoxy, halo, cyano, nitro, trihalomethyl, and thioalkoxy.

The term “aralkyl” refers to alkylene-aryl groups preferably having from1 to 10 carbon atoms in the alkylene moiety and from 6 to 10 carbonatoms in the aryl moiety. Such alkaryl groups are exemplified by benzyl,phenethyl, and the like.

The term “silyl” refers to the group R₃Si—, wherein each R isindependently selected from H, D, C1-20 alkyl, C1-20 deuterated alkyl,fluoroalkyl, aryl, or deuterated aryl. In some embodiments, one or morecarbons in an R alkyl group are replaced with Si.

The term “glycosyl” refers to free radical group obtained by removingthe hemiacetal hydroxyl group from the cyclic form of a monosaccharideor lower oligosaccharide.

The term “polyether” refers to a compound that contains more than oneether group.

The term “solvate” refers to a pharmaceutically acceptable solid form ofa specified compound containing solvent molecules as part of the crystalstructure. A solvate typically retains at least some of the biologicaleffectiveness of such compound. Solvates can have differentsolubilities, hygroscopicities, stabilities, and other properties.Examples of solvates include, but are not limited to, compounds incombination with water, isopropanol, ethanol, methanol, DMSO, ethylacetate, acetic acid, or ethanolamine. Solvates are sometimes termed“pseudopolymorphs.”

The term “hydrate” refers to a solvate with water.

The term “racemate” refers to a mixture that contains an equal amount ofenantiomers.

It will be appreciated by one of ordinary skill in the art thatasymmetric centers may exist in any of the compounds disclosed herein.Thus, the compounds and pharmaceutical compositions thereof may be inthe form of an individual enantiomer, diastereomer, or geometric isomer,or may be in the form of a mixture of stereoisomers. In certainembodiments, the compounds are enantiopure compounds. In certain otherembodiments, mixtures of stereoisomers or diastereomers are provided.Additionally, the compounds encompass both (Z) and (E) double bondisomers (or cis and trans isomers) unless otherwise specificallydesignated. Thus, compounds generally depicted in structures hereinencompass those structures in which double bonds are (Z) or (E).

It will be appreciated that any of the compounds described herein may besubstituted with any number of substituents or functional moieties. Ingeneral, the term “substituted” whether preceded by the term“optionally” or not, and substituents contained in formulas, refer tothe replacement of hydrogen atoms in a given structure with a specifiedsubstituent. When more than one position in any given structure may besubstituted with more than one substituent selected from a specifiedgroup, the substituent may be either the same or different at everyposition.

As used herein, the term “substituted” is contemplated to include allpermissible substituents of organic compounds. In a broad aspect, thepermissible substituents include acyclic and cyclic, branched andunbranched, carbocyclic and heterocyclic, aromatic and nonaromaticsubstituents or organic compounds. For purposes of explanation herein,heteroatoms such as nitrogen may have hydrogen substituents and/or anypermissible substituents of organic compounds described herein whichsatisfy the valencies of the heteroatoms. Furthermore, there is not anyintention to be limited in any manner by the permissible substituents ororganic compounds. Combinations of substituents and variables envisionedare preferably those that result in the formation of stable compoundsuseful in the treatment, for example, of proliferative disordersincluding, but not limited to, cancer.

The term “stable” as used herein refers to compounds which possessstability sufficient to allow manufacture and which maintain theintegrity of the compound for a sufficient period of time to be detectedand preferably for a sufficient period of time to be useful for thepurposes detailed herein.

The term “therapeutically effective amount” as used herein means thatamount of active compound or pharmaceutical agent that elicits thebiological or medicinal response in a tissue system, animal, or humanthat is being sought by a researcher, veterinarian, medical doctor, orother clinician, which includes alleviation of the symptoms of thedisease or disorder being treated.

The term “pharmaceutically acceptable salt” means a salt of a compound.Suitable pharmaceutically acceptable salts of the compounds include acidaddition salts which may, for example, be formed by mixing a solution ofthe compound with a solution of a pharmaceutically acceptable acid.

The term “pharmaceutically acceptable carrier” means a medium that isused to prepare a desired dosage form of the compound. Apharmaceutically acceptable carrier includes solvents, diluents, orother liquid vehicles; dispersion or suspension aids; surface activeagents; isotonic agents; thickening or emulsifying agents;preservatives; solid binders; lubricants; and the like.

General Description

Described herein is a class of small drug-like molecules which havesignificant selective toxicity toward the NSCLC cell line NCI-H522 andclinical application in the treatment of many diseases such as, forexample, non-small cell lung cancer, melanoma, ovarian cancer, CNScancer, and renal cancer. Certain embodiments of these molecules rapidlykill cells in vitro, and show significant selectivity and toxicity to asubset of cell lines tested including melanoma LOX IMVI, ovarian cancerIGROV1, and renal cancer UO-31.

Described herein are compounds that are analogues of2-methyl-3-(thiazol-4-yl)cyclopent-2-enol and3-(2-ethynylthiazol-4-yl)-2methylcyclopent-2-enone, the compounds havingterminal alkyne substituents at the 2-position of the thiazole ring. Thecompounds have thiazole and other aromatic ring systems with terminalalkyne groups. The thiazole substituents other than the terminal alkynegroups can be of varying hydrophobicity, hydrophilicity, and size.

In a broad aspect, the compounds of the present disclosure have thefollowing structural formulas I or II:

and salts, stereoisomers, solvates, and hydrates thereof, where for bothstructures n=1 or 2; X is N or CH; Y is S, O, or NH; R is ═O; 2H; —OR₃,wherein R₃ is hydrogen, alkyl, aryl, glycosyl, or polyether groups;(CO)OR₄, wherein R₄ is hydrogen, alkyl, aryl, or aralkyl groups; —NR₅R₆,wherein R₅ and R₆ are H, alkyl, aryl, or aralkyl groups; or ═N—OR₇,wherein R₇ is hydrogen, alkyl, aryl, aralkyl, glycosyl, or polyethergroups; R₁ is hydrogen, alkyl-, aryl-, or aralkyl; Z is absent orselected from the group consisting of: halide, wherein R₂ is thenabsent; oxygen; nitrogen, wherein there are then two R₂ groups; (CO)O—;O(CO)—; O(CO)O—; (CO)N<, wherein there are then two R₂ groups; NH(CO)—;and, NH(CO)N<, wherein there are then two R₂ groups; wherein when Z isabsent, R₂ connects directly to the structure; and R₂ is hydrogen,alkyl-, aryl-, aralkyl-, glycosyl-, or polyether-, wherein multipleoccurrences of R₂ may be the same or different.

Some of the crystalline forms for the compounds may exist as polymorphsand as such are included. In addition, some of the compounds herein mayform solvates with water (i.e., hydrates) or common organic solvents,which are also included.

Protected forms of the compounds herein are further provided herein. Avariety of protecting groups are possible. Suitable protecting groupsinclude, but are not limited to: benzyl, t-butyl dimethyl silyl,isobutyryl, acetyl, phenoxyacetyl, allyloxycarbonyl (AOC),diisobutylformamidine, benzoyl, formyl, trifluoroacetyl,benzyloxycarbonyl (Cbz), substituted benzyloxycarbonyl, dimethoxy trityl(DMT), monomethoxytrityl (MMT), 9-fluorenylmethyloxycarbonyl (Fmoc),t-butyloxycarbonyl (Boc), isopropyloxycarbonyl, cyclohexyloxycarbonyl,and combinations thereof. By way of non-limiting example, ahydroxyl-protected form of the compounds are those where at least one ofthe hydroxyl groups is protected by a hydroxyl protecting group.Suitable hydroxyl protecting groups include, but are not limited to:alkyl silyl groups such as trimethylsilyl or tert-butyldimethylsilyl;alkyl ethers such as tetrahydropyranyl or tert-butyl; and esters such asacetate.

Prodrugs of the compounds are further provided. In general, suchprodrugs are functional derivatives of the compounds that are readilyconvertible in vivo into the required compound. Thus, in the methods oftreatment, the term “administering” includes the treatment of thevarious disorders described with the compound specifically disclosed orwith a compound which may not be specifically disclosed, but whichconverts to the specified compound in vivo after administration to asubject in need thereof.

In certain embodiments, the compounds have the following structuralformulas:

Compound 2 (having a structural formula referred to herein as FormulaIII), an alcohol, shares a common 4-cyclopentenyl-2-ethynylthiazoleskeleton with compound 3 (which shares a general structural formula withcompound 4, the general structural formula of compounds 3 and 4 alsoreferred to herein as Formula IV), a ketone. For ease of reference,compounds 2, 3, and 4 may therefore be referred to as CETZOLE compounds,or CETZOLEs. A non-limiting scheme of the synthesis of CETZOLEs 2 and 3is shown in FIG. 1. As shown in FIG. 1, Sonogashira coupling of thedibromothiazole 5 with TMS-acetylene gives 6, which can be subjected toStille coupling with the iodoalkene 8 to give 9. Desilylation of 9 withpotassium carbonate in methanol gives compound 3, which can be subjectedto stereoselective CBS reduction to give the alcohol 2. Thestereochemistry of 2 can be confirmed using, for example, the Mosherester model. A non-limiting scheme of the synthesis of CETZOLE 4 isshown in FIG. 6. As shown in FIG. 6, a method of synthesizing CETZOLE 4,which is the α-benzyl derivative of 3, involves treating compound 3 withlithium diisopropylamide (LDA) and benzyl bromide, then desilylating theproduct.

In certain embodiments, the compounds described herein show significantselectivity towards NCI-H522 cells, and are toxic only to a subset ofother cell lines tested, thus making these compounds useful for thetreatment of NSCLC. In certain embodiments, the compounds kill cellsmore rapidly and efficiently than the classical drugs adriamycin,hydroxyurea, and taxol®. Without wishing to be bound by theory, thecompounds do not overtly alter tubulin or block cells in mitosis.Rather, the compounds kill cells in a manner other than by apoptosisbased on the fact that these drugs induce neither membrane blebbing nornuclear condensation, both of which are hallmarks of apoptosis. Againwithout wishing to be bound by theory, it is believed the compounds havean iron-dependent mechanism of action. Thus, described herein is amethod of treating mammalian diseases characterized by the undesirableproliferation of cells, the method comprising administering an agentthat is lethal to the cells through mechanisms that do not depend onapoptosis, wherein the agent comprises a compound having a4-cyclopentyenyl-2-ethynylthiazole skeleton and a terminal alkyne at the2-position of the thiazole ring.

In certain embodiments, the toxicity of the CETZOLE compounds iselevated in cells with activation of the RAS oncogene pathway.Activating mutations in RAS are common in many types of cancer (e.g.,about 15% of lung cancer and about 70% of pancreatic cancer).Accordingly, in certain embodiments, the compounds of the presentdisclosure exhibit outstanding clinical specificity. In certainembodiments, the toxicity of CETZOLE compounds is also elevated in cellslacking E-cadherin, which exhibit a mesenchymal phenotype. Themesenchymal/Ecadherin^(minus) phenotype is associated with stem cells ofvarious solid tumors. The selective killing of mesenchymal cellsprovides a way to kill cancer stem cells, thereby reducing metastaticspread and tumor relapse after therapy.

Pharmaceutical Compositions

The compounds described herein can be incorporated into pharmaceuticalcompositions for use in the treatment of various diseases or disorders.In certain embodiments, CETZOLE compounds 2, 3, or 4 are especiallyuseful in pharmaceutical compositions for the treatment of non-smallcell lung cancer, ovarian cancer, renal cancer, CNS cancer, or melanoma.

A pharmaceutical composition as described herein may be formulated withany of the compounds disclosed herein, plus any common excipients,diluents, or carriers. The compositions can be compressed into tablets,or formulated as elixirs or solutions for convenient oral administrationor administration by intramuscular or intravenous routes. The compoundscan be administered transdermally, may be formulated as sustainedrelease dosage forms, and the like.

The compounds, compositions, and formulations provided herein are usefulfor treating animals, such as humans, for various diseases. A method oftreating a human patient according to the present disclosure includesthe administration of an effective amount of a CETZOLE compounddescribed herein or pharmaceutical composition comprising a CETZOLEcompound. The CETZOLE compounds can be formulated into compositionswhich may be administered by the oral and rectal routes, topically,parenterally, e.g., by injection, and by continuous or discontinuousintra-arterial infusion, in the form of, for example, tablets, lozenges,sublingual tablets, sachets, cachets, elixirs, gels, suspensions,aerosols, ointments, for example, containing from 1 to 10% by weight ofthe active compound in a suitable base, soft and hard gelatin capsules,suppositories, injectable solutions, and suspensions in physiologicallyacceptable media, and sterile packaged powders adsorbed onto a supportmaterial for making injectable solutions. Advantageously for thispurpose, compositions may be provided in dosage unit form, preferablyeach dosage unit containing from about 5 to about 500 mg (from about 5to about 50 mg in the case of parenteral or inhalation administration,and from about 25 to about 500 mg in the case of oral or rectaladministration) the compounds. Dosages from about 0.5 to about 300 mg/kgper day, preferably 0.5 to 20 mg/kg, of active ingredient may beadministered although it will, of course, readily be understood that theamount of the compound actually to be administered will be determined bya physician, in light of all the relevant circumstances including thecondition to be treated, the choice of compound to be administered, andthe choice of route of administration. Therefore, the dosage rangesdiscussed herein are not intended to limit the scope of the presentinvention in any way.

The formulations useful for separate administration of the CETZOLEcompounds normally contain at least one CETZOLE compound (which may bereferred to herein as the active ingredient or active substance) mixedwith a carrier, or diluted by a carrier, or enclosed or encapsulated byan ingestible carrier in the form of a capsule, sachet, cachet, paper,or other container, or by a disposable container such as an ampoule. Acarrier or diluent may be a solid, semi-solid, or liquid material whichserves as a vehicle, excipient, or medium for the active therapeuticsubstance. Some examples of the diluents or carrier which may beemployed in the pharmaceutical compositions of the present disclosureare lactose, dextrose, sucrose, sorbitol, mannitol, propylene glycol,liquid paraffin, white soft paraffin, kaolin, fumed silicon dioxide,microcrystalline cellulose, calcium silicate, silica,polyvinylpyrrolidone, cetostearyl alcohol, starch, modified starches,gum acacia, calcium phosphate, cocoa butter, ethoxylated esters, oil oftheobroma, arachis oil, alginates, tragacanth, gelatin, syrup, methylcellulose, polyoxyethylene sorbitan monolaurate, ethyl lactate, methyland propyl hydroxybenzoate, sorbitan trioleate, sorbitan sesquioleateand oleyl alcohol, and propellants such as trichloromonofluoromethane,dichlorodifluoromethane, and dichlorotetrafluoroethane. In the case oftablets, a lubricant may be incorporated to prevent sticking and bindingof the powdered ingredients in the dies and on the punch of thetableting machine. For such purpose there may be employed, for instance,aluminum, magnesium, or calcium stearates, talc, or mineral oil.

In certain embodiments, pharmaceutical compositions of the presentdisclosure comprise an effective amount of a CETZOLE compounds and/oradditional agents, dissolved or dispersed in a pharmaceuticallyacceptable carrier. The phrases “pharmaceutical” or “pharmacologicallyacceptable” refers to molecular entities and compositions that produceno adverse, allergic, or other untoward reaction when administered to ananimal, such as, for example, a human. The preparation of apharmaceutical composition that contains at least one compound oradditional active ingredient will be known to those of skill in the artin light of the present disclosure, as exemplified by Remington'sPharmaceutical Sciences, 2003, incorporated herein by reference.Moreover, for animal (e.g., human) administration, it will be understoodthat preparations should meet sterility, pyrogenicity, and generalsafety and purity standards as required by FDA Office of BiologicalStandards.

A composition disclosed herein may comprise different types of carriersdepending on whether it is to be administered in solid, liquid, oraerosol form, and whether it needs to be sterile for such routes ofadministration as injection. Compositions disclosed herein can beadministered intravenously, intradermally, transdermally, intrathecally,intraarterially, intraperitoneally, intranasally, intravaginally,intrarectally, intraosseously, periprosthetically, topically,intramuscularly, subcutaneously, mucosally, in utero, orally, topically,locally, via inhalation (e.g., aerosol inhalation), by injection, byinfusion, by continuous infusion, by localized perfusion bathing targetcells directly, via a catheter, via a lavage, in cremes, in lipidcompositions (e.g., liposomes), or by other method or any combination ofthe forgoing as would be known to one of ordinary skill in the art.

The actual dosage amount of a composition disclosed herein administeredto an animal or human patient can be determined by physical andphysiological factors such as body weight, severity of condition, thetype of disease being treated, previous or concurrent therapeuticinterventions, idiopathy of the patient and on the route ofadministration. Depending upon the dosage and the route ofadministration, the number of administrations of a preferred dosageand/or an effective amount may vary according to the response of thesubject. The compounds of the present disclosure are generally effectiveover a wide dosage range. The practitioner responsible foradministration will, in any event, determine the concentration of activeingredient(s) in a composition and appropriate dose(s) for theindividual subject.

In certain embodiments, pharmaceutical compositions may comprise, forexample, at least about 0.1% of an active compound. In otherembodiments, an active compound may comprise between about 2% to about75% of the weight of the unit, or between about 25% to about 60%, forexample, and any range derivable therein. Naturally, the amount ofactive compound(s) in each therapeutically useful composition may beprepared in such a way that a suitable dosage will be obtained in anygiven unit dose of the compound. Factors such as solubility,bioavailability, biological half-life, route of administration, productshelf life, as well as other pharmacological considerations, will becontemplated by those preparing such pharmaceutical formulations, and assuch, a variety of dosages and treatment regimens may be desirable.

In other non-limiting examples, a dose may also comprise from about 1microgram/kg/body weight, about 5 microgram/kg/body weight, about 10microgram/kg/body weight, about 50 microgram/kg/body weight, about 100microgram/kg/body weight, about 200 microgram/kg/body weight, about 350microgram/kg/body weight, about 500 microgram/kg/body weight, about 1milligram/kg/body weight, about 5 milligram/kg/body weight, about 10milligram/kg/body weight, about 50 milligram/kg/body weight, about 100milligram/kg/body weight, about 200 milligram/kg/body weight, about 350milligram/kg/body weight, about 500 milligram/kg/body weight, to about1000 mg/kg/body weight or more per administration, and any rangederivable therein. In non-limiting examples of a derivable range fromthe numbers listed herein, a range of about 5 mg/kg/body weight to about100 mg/kg/body weight, about 5 microgram/kg/body weight to about 500milligram/kg/body weight, etc., can be administered, based on thenumbers described above.

In certain embodiments, a composition and/or additional agent isformulated to be administered via an alimentary route. Alimentary routesinclude all possible routes of administration in which the compositionis in direct contact with the alimentary tract. Specifically, thepharmaceutical compositions disclosed herein may be administered orally,buccally, rectally, or sublingually. As such, these compositions may beformulated with an inert diluent or with an assimilable edible carrier,or they may be enclosed in hard- or soft-shell gelatin capsules, or theymay be compressed into tablets, or they may be incorporated directlywith the food of the diet.

In further embodiments, a composition described herein may beadministered via a parenteral route. As used herein, the term“parenteral” includes routes that bypass the alimentary tract.Specifically, the pharmaceutical compositions disclosed herein may beadministered, for example but not limited to, intravenously,intradermally, intramuscularly, intraarterially, intrathecally,subcutaneous, or intraperitoneally (U.S. Pat. Nos. 6,753,514; 6,613,308;5,466,468; 5,543,158; 5,641,515; and 5,399,363 are each specificallyincorporated herein by reference in their entirety).

Solutions of the compositions disclosed herein as free bases orpharmacologically acceptable salts may be prepared in water suitablymixed with a surfactant, such as hydroxypropylcellulose. Dispersions mayalso be prepared in glycerol, liquid polyethylene glycols, and mixturesthereof, and in oils. Under ordinary conditions of storage and use,these preparations contain a preservative to prevent the growth ofmicroorganisms. The pharmaceutical forms suitable for injectable useinclude sterile aqueous solutions or dispersions and sterile powders forthe extemporaneous preparation of sterile injectable solutions ordispersions (U.S. Pat. No. 5,466,468, specifically incorporated hereinby reference in its entirety). In all cases the form should be sterileand should be fluid to the extent that easy injectability exists. Itshould be stable under the conditions of manufacture and storage andshould be preserved against the contaminating action of microorganisms,such as bacteria and fungi. The carrier can be a solvent or dispersionmedium containing, for example, water, ethanol, a polyol (i.e.,glycerol, propylene glycol, and liquid polyethylene glycol, and thelike), suitable mixtures thereof, and/or vegetable oils. Proper fluiditymay be maintained, for example, by the use of a coating, such aslecithin, by the maintenance of the required particle size in the caseof dispersion, and by the use of surfactants. The prevention of theaction of microorganisms can be brought about by various antibacterialand antifungal agents, for example, parabens, chlorobutanol, phenol,sorbic acid, thimerosal, and the like. In many cases, it is preferableto include isotonic agents, such as, but not limited to, sugars orsodium chloride. Prolonged absorption of the injectable compositions canbe brought about by the use in the compositions of agents delayingabsorption such as, for example, aluminum monostearate, or gelatin.

For parenteral administration in an aqueous solution, for example, thesolution should be suitably buffered if necessary and the liquid diluentfirst rendered isotonic with sufficient saline or glucose. Theseparticular aqueous solutions are especially suitable for intravenous,intramuscular, subcutaneous, and intraperitoneal administration. Sterileaqueous media that can be employed will be known to those of skill inthe art in light of the present disclosure. For example, one dosage maybe dissolved in 1 mL of isotonic NaCl solution and either added to 1000mL of hypodermoclysis fluid or injected at the proposed site ofinfusion, (see for example, “Remington's Pharmaceutical Sciences” 15thEdition, pages 1035-1038 and 1570-1580). Some variation in dosage willnecessarily occur depending on the condition of the subject beingtreated. The person responsible for administration will, in any event,determine the appropriate dose for the individual subject.

Sterile injectable solutions are prepared by incorporating thecompositions in the required amount in the appropriate solvent withvarious other ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the various sterilized compositions into a sterile vehiclewhich contains the basic dispersion medium and the required otheringredients from those enumerated above. In the case of sterile powdersfor the preparation of sterile injectable solutions, some methods ofpreparation are vacuum-drying and freeze-drying techniques which yield apowder of the active ingredient plus any additional desired ingredientfrom a previously sterile-filtered solution thereof. A powderedcomposition is combined with a liquid carrier such as, e.g., water or asaline solution, with or without a stabilizing agent.

In other embodiments, the compositions may be formulated foradministration via various miscellaneous routes, for example, topical(i.e., transdermal) administration, mucosal administration (intranasal,vaginal, etc.) and/or via inhalation. Pharmaceutical compositions fortopical administration may include the compositions formulated for amedicated application such as an ointment, paste, cream or powder.Ointments include all oleaginous, adsorption, emulsion, andwater-soluble based compositions for topical application, while creamsand lotions are those compositions that include an emulsion base only.Topically administered medications may contain a penetration enhancer tofacilitate adsorption of the active ingredients through the skin.Suitable penetration enhancers include glycerin, alcohols, alkyl methylsulfoxides, pyrrolidones, and luarocapram. Possible bases forcompositions for topical application include polyethylene glycol,lanolin, cold cream, and petrolatum as well as any other suitableabsorption, emulsion, or water-soluble ointment base. Topicalpreparations may also include emulsifiers, gelling agents, andantimicrobial preservatives as necessary to preserve the composition andprovide for a homogenous mixture. Transdermal administration of thecompositions may also comprise the use of a “patch.” For example, thepatch may supply one or more compositions at a predetermined rate and ina continuous manner over a fixed period of time.

In certain embodiments, the composition is in a form which is suitablefor application to human skin. In certain embodiments, the compositionis in the form of an oil, ointment, cream, lotion, or gel. In certainembodiments, the composition contains, as additional ingredients, anyof: water, oil, alcohols (such as ethanol, isopropanol, or propanol),emulsifying agents, perfumes, coloring agents, fillers, abrasive agents,moisturizers, or combinations thereof.

In certain embodiments, the compositions are suitable for delivery byeye drops, intranasal sprays, inhalation, and/or other aerosol deliveryvehicles. Methods for delivering compositions directly to the lungs vianasal aerosol sprays have been described in U.S. Pat. No. 5,756,353 and5,804,212 (each specifically incorporated herein by reference in theirentirety). Likewise, the delivery of drugs using intranasalmicroparticle resins (Takenaga et al., 1998) andlysophosphatidyl-glycerol compounds (U.S. Pat. No. 5,725, 871,specifically incorporated herein by reference in its entirety) are alsowell-known in the pharmaceutical arts and could be employed to deliverthe compositions described herein. Likewise, transmucosal drug deliveryin the form of a polytetrafluoroetheylene support matrix is described inU.S. Pat. No. 5,780,045 (specifically incorporated herein by referencein its entirety), and could be employed to deliver the compositionsdescribed herein.

It is further envisioned the compositions disclosed herein may bedelivered via an aerosol. The term aerosol refers to a colloidal systemof finely divided solid or liquid particles dispersed in a liquefied orpressurized gas propellant. The typical aerosol for inhalation consistsof a suspension of active ingredients in liquid propellant or a mixtureof liquid propellant and a suitable solvent. Suitable propellantsinclude hydrocarbons and hydrocarbon ethers. Suitable containers willvary according to the pressure requirements of the propellant.Administration of the aerosol will vary according to subject's age,weight, and the severity and response of the symptoms.

In a composition, the CETZOLE compound may be in free form or, whereappropriate, as pharmaceutically acceptable derivatives such asprodrugs, salts, and/or esters of the compound. The composition may bein any suitable form such as solid, semisolid, or liquid form. Ingeneral, the pharmaceutical preparation will contain one or more of thecompounds as an active ingredient in an admixture with an organic orinorganic carrier or excipient suitable for external, enteral, orparenteral application. The active ingredient may be compounded, forexample, with the usual non-toxic, pharmaceutically acceptable carriersfor tablets, pellets, capsules, suppositories, pessaries, solutions,emulsions, suspensions, and any other form suitable for use. Thecarriers that can be used include water, glucose, lactose, gum acacia,gelatin, mannitol, starch paste, magnesium trisilicate, talc,cornstarch, keratin, colloidal silica, potato starch, urea, and othercarriers suitable for use in manufacturing preparations, in solid,semi-solid, or liquefied form. In addition, auxiliary stabilizing,thickening, and coloring agents may be used.

Pharmaceutical preparations can include at least one of the compounds asdescribed herein, or a pharmaceutically acceptable derivative thereof,which compounds are capable of inhibiting the growth of or killingcancer cells, and, in certain embodiments, are capable of inhibiting thegrowth of or killing multidrug-resistant cancer cells. In certainembodiments, the pharmaceutical preparations also comprise asolubilizing or emulsifying agent.

In one aspect, the compounds and compositions are used to treat cancerssuch as non-small cell lung cancer, renal cancer, ovarian cancer, CNScancer, and melanoma. The method of treatment comprises administering atherapeutically effective amount of a compound to a subject sufferingfrom cancer or other disease. The method may be repeated as necessaryeither to mitigate (i.e., prevent further growth) or to eliminate thecancer. Clinically, practice of the method will result in a reduction inthe size or number of the cancerous growth and/or a reduction inassociated symptoms (where applicable). Pathologically, practice of themethod will produce at least one of the following: inhibition of cancercell proliferation, reduction in the size of the cancer or tumor,prevention of further metastasis, and inhibition of tumor angiogensis.

The compounds and compositions can be used in combination therapies.That is, the compounds and compositions can be administered concurrentlywith, prior to, or subsequent to one or more other desired therapeuticor medical procedures or drugs. The particular combination of therapiesand procedures in the combination regimen will take into accountcompatibility of the therapies and/or procedures and the desiredtherapeutic effect to be achieved. Combination therapies includesequential, simultaneous, and separate administration of the activecompound in a way that the therapeutic effects of the first administeredprocedure or drug is not entirely disappeared when the subsequentprocedure or drug is administered.

By way of a non-limiting example of a combination therapy, the CETZOLEcompounds or compositions can be administered in combination with one ormore suitable anti-cancer agents including, but not limited to:chemotherapeutic agents; cytotoxins; antimetabolites; alkylating agents;protein kinase inhibitors; anthracyclines; antibiotics; antimitoticagents (e.g. antitubulin agents); corticosteroids; radiopharmaceuticals;proteins such as cytokines, enzymes, or interferons; biological responsemodifiers such as krestin, lentinan, sizofiran, picibanil, ubenimex;anti-angiogenic compounds such as acitretin, fenretinide, thalidomide,zoledronic acid, angiostatin, aplidine, cilengtide, combretastatin A-4,endostatin, halofuginone, rebimastat, removab®, Revlimid®, squalamine,ukrain, or Vitaxin®; platinum-coordinated compounds such as cisplatin,carboplatin, nedaplatin, or oxaliplatin; camptothecin derivatives suchas camptothecin, 10-hydroxycamptothecin, 9-aminocamptothecin,irinotecan, SN-38, edotecarin, or topotecan; compounds or chelates thatinclude radionuclides; or combinations thereof. Examples of suitableinterferons include, but are not limited to interferon alpha, interferonalpha-2a, interferon, alpha-2b, interferon beta, interferon gamma-1a,interferon gamma-1b (Actimmune), interferon gamma-n1, or combinationsthereof.

In certain embodiments, the anti-cancer agent is one or more ofhydroxyureas, taxol®, adriamycin, 5-fluorouracil, cyclophosphamide.etoposide, altretamine, ifosfamide, vinblastine sulfate, estramustinephosphate, suramin, strontium-89, filgrastim, lentinan, sizofilan,TheraCys®, ubenimex, WF-10, aldesleukin, alemtuzumab, BAM-002,dacarbazine, daclizumab, denileukin, gemtuzumab ozogamicin, ibritumomab,imiquimod, lenograstim, lentinan, Corixa, molgramostim, OncoVAX-CL,sargramostim, tasonermin, tecleukin, thymalasin, tositumomab,Virulizin®, Z-100, epratuzumab, mitumomab, oregovomab, pemtumomab(Y-muHMFGI), Provenge® (Dendreon), alitretinoin, ampligen, atrasentanbexarotene, bortezomib, bosentan, calcitriol, exisulind, finasteride,fotemustine, ibandronic acid, miltefosine, mitoxantrone, 1-asparaginase,procarbazine, dacarbazine, hydroxycarbamide, pegaspargase, pentostatin,tazarotne, Telcyta® (TLK-286, Telik Inc.), Velcade® (bortemazib,Millenium), or tretinoinor.

Another non-limiting example of a combination therapy for NSCLC or othercancers is the combination of a CETZOLE compound or CETZOLE-containingcomposition with one or more surgical treatments. Suitable surgicaltreatments include, but are not limited to, a wedge resection, alobectomy, a pneumonectomy, a sleeve reduction, a hysterectomy, abilaterial salpingo-oophorectomy, an omentectomy, or a nephrectomy.Other possible therapies suitable for combination with a CETZOLEcompound or CETZOLE-containing composition include, but are not limitedto, immunotherapy, hormone therapy, radiation therapy, or a combinationthereof.

Embodiments of the present disclosure further include methods ofdetermining coverage or denial of health insurance reimbursement and/orpayment for treatments of disease comprising the compounds orcompositions described herein. In certain embodiments, the treatmentcomprises a CETZOLE compound or CETZOLE-containing pharmaceuticalcomposition, and a provider of health insurance denies coverage orreimbursement for the treatment.

Kits

The compounds or methods described herein could be embodied as parts ofa kit or kits. A non-limiting example of such a kit comprises one ormore CETZOLE compounds and a pharmaceutically acceptable carrier,diluent, or excipient in separate containers, where the containers mayor may not be present in a combined configuration. Many other kits arepossible, such as kits for preparing a CETZOLE compound. In certainembodiments, a kit for preparing a CETZOLE compound comprises adibromothiazole and TMS-acetylene in separate containers. In particularembodiments, the kit further comprises an iodoalkene, lithiumdiisopropylamide, benzyl bromide, or combinations thereof.

The kits may further include instructions for using the components ofthe kit(s) to practice the subject methods. The instructions forpracticing the subject methods are generally recorded on a suitablerecording medium. For example, the instructions may be present in thekits as a package insert or in the labeling of the container of the kitor components thereof. In other embodiments, the instructions arepresent as an electronic storage data file present on a suitablecomputer readable storage medium, such as a flash drive, CD-ROM, ordiskette. In other embodiments, the actual instructions are not presentin the kit, but means for obtaining the instructions from a remotesource, such as via the internet, are provided. An example of thisembodiment is a kit that includes a web address where the instructionscan be viewed and/or from which the instructions can be downloaded. Aswith the instructions, this means for obtaining the instructions isrecorded on a suitable substrate.

EXAMPLES

Synthesis of Compounds 2 and 3

A mixture of commercially available 2,4-dibromothiazole 5 (3.000 g,12.34 mmol, 1 equiv), triphenylphosphine (486 mg, 1.851 mmol, 5 mol %),CuI (120 mg, 0.617 mmol, 5 mol %), and Pd(PPh₃)₂Cl₂ (120 mg, 0.173 mmol,1.4 mol %) was placed in a three-neck roundbottom flask under nitrogen.Anhydrous toluene (42 mL) was added followed by anhydrous Et₃N (2.2 mL,15.04 mmol, 1.3 equiv) and trimethylsilylacetylene (2.6 mL, 18.51 mmol,1.5 equiv). The reaction mixture was refluxed at 140° C. for 2 d. It waspoured into water (50 mL) and extracted with EtOAc (3×20 mL). Thecombined organic extract was dried over anhydrous sodium sulfate andconcentrated in vacuo. The crude product was purified by flashchromatography on silica gel in CH₂Cl₂-hexanes to obtain 6 (2.625 g,81%) as a brownish/red solid: TLC R_(f)=0.72 (10% EtOAc-hexanes); mp 37°C.; IR: 3300, 2900, 2250 cm⁻¹; ¹H NMR (400 MHz, CDCl₃): δ 7.18 (s, 1H),0.24 (s, 9H); ¹³C NMR (CDCl₃, 100 MHz): δ 149.54, 125.87, 118.85,103.03, 95.52, −0.41; HRMS (m/z): [M+H]⁺ calculated for C₈H₁₁NSSiBr,259.9565; found, 259.9565.

A solution of 2-((trimethylsilyl)ethynyl)-4-bromothiazole 6 (100 mg,0.384 mmol, 1 equiv) in anhydrous ether (3 mL) under N₂ was cooled to−78° C. and t-BuLi (240 μL, 1.6 M, 0.786 mmol, 1 equiv) was addeddropwise. The reaction mixture was stirred at -78° C. for 1 hr and asolution of trimethyltin chloride (150 mg, 0.768 mmol, 2 equiv) inanhydrous ether (2 mL) was added via syringe. The reaction mixture wasstirred at −78° C. for an additional 1 hour and was allowed to warm toroom temperature slowly. The reaction mixture was diluted with hexanes,passed through a silica gel pad deactivated with 5% Et₃N-hexanes, elutedwith EtOAc, and concentrated in vacuo to obtain a yellow oil 7, whichwas immediately taken to the next step. ¹H NMR (600 MHz, CDCl₃): δ 7.33(s, 1H), 0.34 (t, J=14.8 Hz, 9H), 0.25 (m, 3H).

3-Iodo-2-methylcyclopent-2-enone 8 (83 mg, 3.84 mmol, 1 equiv) andPd(PPh₃)₄ (88 mg, 0.078 mmol, 20 mol %) were placed in a μw vial underN₂. A solution of tin derivative 7 prepared above in dry DMF (2 mL) wasadded via syringe. The μw vial was heated for 2 hours and 15 minutes ina microwave synthesizer at 145° C. The reaction mixture was poured intowater (30 mL) and extracted with EtOAc (3×, 10 mL). The combined organicextract was dried over anhydrous sodium sulfate and concentrated invacuo. The crude product was purified by flash chromatography on silicagel in EtOAc-hexanes and recrystallized from CH₂Cl₂-hexanes to obtain 9as tan, star-shaped crystals (50 mg, 47%): TLC R_(f)=0.63 (40%EtOAc-hexanes); mp 108-110° C.; IR: 3300, 2900, 2200, 1750 cm⁻¹; ¹H NMR(600 MHz, CDCl₃): δ 7.58 (s, 1H), 2.99 (t, J=2.0 Hz, 2H), 2.52 (m, 2H),2.10 (d, J=2.0 Hz, 3H), 0.28 (s, 9H); ¹³C NMR (CDCl₃, 100 MHz): δ209.71, 157.66, 152.65, 148.22, 137.37, 121.79, 102.05, 96.15, 33.74,28.18, 10.23, −0.46; HRMS (m/z): [M +Na]⁺ calculated for C₁₄H₁₇NOSSi,298.0698; found, 298.0680.

A solution of2-methyl-3-(2-((trimethylsilyl)ethynyl)thiazol-4-yl)cyclopent-2-enone 9(50 mg, 0.182 mmol, 1 equiv) and K₂CO₃ (3 mg, 0.022 mmol, 12 mol %) inmethanol (1 mL) was stirred for 5 minutes. The crude product was pouredinto water (8 mL) and extracted with EtOAc (3×, 3 mL). The combinedorganic extract was dried over anhydrous sodium sulfate and concentratedin vacuo. The product was recrystallized from CH₂Cl₂-hexanes to obtain 3as tan needles: TLC R_(f)=0.33 (40% EtOAc-hexanes); mp 138-139° C.; IR:V_(max) 3000, 2900, 2250, 1750 cm⁻¹; ¹H NMR (400 MHz, CDCl₃): δ 7.62 (s,1H), 3.52 (s, 3H), 2.99-2.96 (m, 2H), 2.53-2.51 (m, 2H), 2.11 (t, J=2.0Hz, 3H); ¹³C NMR (CDCl₃, 100 MHz): δ 209.98, 157.56, 153.08, 147.50,137.84, 121.90, 83.24, 76.31, 33.92, 28.31, 10.38; HRMS (m/z): [M+Na]⁺calculated for C₁₁H₉NOS, 226.0303; found, 226.0298.

A solution of 3-(2-ethynylthiazol-4-yl)-2-methylcyclopent-2-enone 3 (95mg, 0.340, 1 equiv) and R-2-methyl-CBS-oxazaborolidine (19 mg, 0.072mmol, 20 mol %) in anhydrous THF (2 mL) was cooled to 0° C. A solutionof borane-Me₂S (170 μL, 0.340 mmol, 1 equiv) in THF was added dropwise.The reaction mixture was stirred at 0° C. for 25 minutes and thereaction was quenched by slow addition of water (5 mL) at 0° C. Thereaction mixture was extracted with EtOAc (3×2 mL). The combined organicextract was dried over anhydrous sodium sulfate and concentrated invacuo. The crude product was purified by preparative thin-layerchromatography on silica gel in 25% EtOAc-CH₂Cl₂ to obtain the partialpurified product 2 (72 mg, 75.1%) as a reddish/brown solid: TLCR_(f)=0.58 (40% EtOAc-CH₂Cl₂); mp 69-71° C.; [α]²⁵ _(D)=−12.8° (c=1.50,CHCl₃); IR: V_(max) 3300, 2900, 2300 cm⁻¹; ¹H NMR (400 MHz, CDCl₃): δ7.13 (s, 1H), 4.73 (d, J=4.4 Hz, 1H), 3.44 (s, 1H), 2.88-2.81 (m, 1H),2.67-2.60 (m, 1H), 2.43-2.34 (m, 1H), 2.14 (d, J=8.8 Hz, 3H), 1.78-1.70(m, 1H); ¹³C NMR (CDCl₃, 100 MHz): δ 154.15, 146.52, 140.24, 130.83,117.48, 82.30, 82.14, 32.91, 32.67, 13.49; HRMS (m/z): [M+Na]⁺calculated for C₁₁H₁₁NOS, 228.0458; found, 228.0459.

Synthesis of Compound 4

n-BuLi (333 μL, 0.841 mmol, 1.2 equiv) was added to a solution ofanhydrous diisopropyl amine (120 μL, 0.841 mmol, 1.2 equiv) in anhydrousTHF (3 mL) at −78° C. The reaction mixture was stirred at −78° C. for 5min and then stirred at 0° C. for 30 minutes. A solution of2-methyl-3-(2-((trimethylsilyl)ethynyl)thiazol-4-yl)cyclopent-2-enone 9(193 mg, 0.700 mmol, 1 equiv) in anhydrous THF (3 mL) was added. Thereaction mixture was stirred for 10 min at 0° C., warmed to roomtemperature, was stirred for 10 min, and benzyl bromide (180 μL, 1.54mmol, 2.2 equiv) was added. The reaction mixture was stirred at roomtemperature overnight. It was then poured into water (10 mL) andextracted with ethyl acetate (3×5 mL). The combined organic extract wasdried over anhydrous sodium sulfate, and the solvent was removed invacuo. The product was purified by flash chromatography on silica gel inEtOAc-hexanes followed by preparative thin-layer chromatography onsilica gel in 20% EtOAc-hexanes to obtain the dibenzylated product andthe monobenzylated product 10: (3.9 mg), mp 153-155° C.; ¹H NMR (400MHz, CDCl₃): δ 7.56 (s, 1H), 7.30-7.26 (m, 2H), 7.23-7.19 (m, 3H), 3.36(dd, J=14.0 Hz, 4.0 Hz, 1H), 3.03-3.01 (m, 1H), 2.86-2.83 (m, 1H),2.73-2.68 (m, 1H), 2.60-2.54 (m, 1H), 2.14 (t, J=2.0 Hz, 3H), 0.28 (t,J=3.6 Hz, 9H); ¹³C NMR (CDCl₃, 100 MHz) δ 210.94, 156.79, 148.40,139.82, 136.68, 129.07, 128.68, 126.51, 122.06, 102.33, 96.18, 46.33,37.58, 434.85, 10.56, −0.31.

A mixture of5-benzyl-2-methyl-3-(2-((trimethylsilyl)ethynyl)thiazol-4-yl)cyclopent-2-enone10 (3.9 mg, 0.11 mmol, 1.0 equiv), K₂CO₃ (0.26 mg, 0.002 mmol, 18 mol %)and MeOH (100 μL) was stirred at room temperature for 10 min. Water (500μL) was added and the mixture was extracted with EtOAc (3×500 μL). Thecombined organic extract was concentrated in vacuo and the product waspurified by preparative thin-layer chromatography on silica gel in 20%EtOAc-hexanes to obtain 4 as a yellow solid (2.3 mg); ¹H NMR (400 MHz,CDCl₃): δ 7.54 (s, 1H), 7.29-7.25 (m, 2H), 7.22-7.17 (m, 3H), 3.50 (s,1H), 3.33 (dd, J=14.0 Hz, 4.4 Hz, 1H), 3.05-2.99 (m, 1H), 2.86-2.80 (m,1H), 2.71-2.65 (m, 1H), 2.59 (dd, J=10.4 Hz, 3.2 Hz, 1H), 2.13 (t, J=2.0Hz, 3H); ¹³C NMR (CDCl₃, 100 MHz) δ 210.86, 156.36, 152.98, 147.47,139.78, 136.94, 130.12, 128.62, 126.53, 122.12, 83.22, 76.27, 46.30,37.56, 34.72, 10.51.

Biological Activity and Selectivity

The compounds described herein were evaluated in comparison to thefollowing open-chain epothilones:

From these compounds, 1f was lethal against non-small cell lung cancercell lines HOP62 (75%) and NCI-H522 (62%), CNS cancer U251 (25%),melanoma LOX IMVI (23%), ovarian cancer IGROV1 (32%), and renal cancerUO-31 (36%). NCI-H522 cells treated with 1f at 10 μM were killedcompletely after 18 hours of treatment. Compound 1f killed NCI-H522cells with an LD₅₀ of 1 μM—more than 20 times lower than the estimatedLD₅₀ for HOP62 cells. However, compounds 1a, 1b, 1c, and 1d had littleeffect at 10 μM even after several days of treatment. The cytotoxiceffect of 1f is due to the alcohol 2 formed by in situ hydrolysis of theester 1f. FIG. 4 displays the effects of compounds 1a, 1c, 1f and 2 oncell viability, in comparison with the drugs hydroxyureas, taxol®, andadriamycin. FIGS. 5A-5C show the effects of 1f on NCI-H522 cells at 10μM.

The effects of compound 2 on NCI-H522 cells, other cancer cells, andnormal cell lines were evaluated. Compound 2 killed NCI-H522 cells at 10μM. Cell staining showed compound 2 had no overt effects on tubulin ininterphase or mitosis, thus showing that compound 2 does not mimicepothilones in mechanism of action. FIGS. 3A-3C show the effects ofcompound 2 on tubulin and mitochondrial function. The rapid mode of celldeath shows compound 2 could disrupt mitochondrial function. However,membrane potentials were intact until late in the death process.

The ketone 3, which is a precursor of 2, was also lethal to NCI-H522cells and has a similar selectivity profile. Compound 4, which is theα-benzyl derivative of 3, was synthesized by treating the ketone 3 withLDA followed by benzyl bromide, and desilylating the product to obtain4, as depicted in FIG. 6. The α-benzylation did not compromise compound4's activity on the NI-H522 cell line, thus providing a SAR neutral sitefor chemical modification. The effects of compounds 2, 3, and 4 onvarious cell lines are shown in FIG. 2.

Certain embodiments of the compounds disclosed herein are defined in theexamples herein. It should be understood that these examples, whileindicating particular embodiments of the invention, are given by way ofillustration only. From the above discussion and these examples, oneskilled in the art can ascertain the essential characteristics of thisinvention, and without departing from the spirit and scope thereof, canmake various changes and modifications of the invention to adapt it tovarious usages and conditions.

While the invention has been described with reference to various andpreferred embodiments, it should be understood by those skilled in theart that various changes may be made and equivalents may be substitutedfor elements thereof without departing from the essential scope of theinvention. In addition, many modifications may be made to adapt aparticular situation or material to the teachings of the inventionwithout departing from the essential scope thereof. Therefore, it isintended that the invention not be limited to the particular embodimentdisclosed herein contemplated for carrying out this invention, but thatthe invention will include all embodiments falling within the scope ofthe claims.

1. A compound comprising the following structural formula:

wherein: n=1 or 2; X is N or CH; Y is S, O, or NH; R is ═O; 2H; —OR₃,wherein R₃ is hydrogen, alkyl, aryl, glycosyl, or polyether groups;(CO)OR₄, wherein R₄ is hydrogen, alkyl, aryl, or aralkyl groups; —NR₅R₆,wherein R₅ and R₆ are H, alkyl, aryl, or aralkyl groups; or ═N—OR₇,wherein R₇ is hydrogen, alkyl, aryl, aralkyl, glycosyl, or polyethergroups; R₁ is hydrogen, alkyl-, aryl-, or aralkyl; Z is absent orselected from the group consisting of: halide, wherein R₂ is absent;oxygen; nitrogen, wherein there are two R₂ groups; (CO)O—; O(CO)—;O(CO)O—; (CO)N<, wherein there are two R₂ groups; NH(CO)—; and NH(CO)N<,wherein there are two R₂ groups; wherein when Z is absent, R₂ connectsdirectly to a ring; and R₂ is hydrogen, alkyl-, aryl-, aralkyl-,glycosyl-, or polyether-, wherein multiple occurrences of R₂ are thesame or different; and prodrugs, salts, stereoisomers, racemates,prodrugs, solvates, and hydrates thereof.
 2. A compound of claim 1having the following structural formula:

and salts, stereoisomers, racemates, prodrugs, solvates, and hydratesthereof.
 3. A compound of claim 1 having the following structuralformula:

wherein R is H, C₆H₅CH₂, or polyether; and salts, stereoisomers,racemates, prodrugs, solvates, and hydrates thereof.
 4. A compound ofclaim 1, further comprising a protecting group.
 5. A compound of claim4, wherein the protecting group is selected from the group consistingof: benzyl, t-butyl dimethyl silyl, isobutyryl, acetyl, phenoxyacetyl,allyloxycarbonyl (AOC), diisobutylformamidine, benzoyl, formyl,trifluoroacetyl, benzyloxycarbonyl (Cbz), substituted benzyloxycarbonyl,dimethoxy trityl (DMT), monomethoxytrityl (MMT),9-fluorenylmethyloxycarbonyl (Fmoc), t-butyloxycarbonyl (Boc),isopropyloxycarbonyl, cyclohexyloxycarbonyl, and a combination thereof.6. A compound of claim 4, wherein the protecting group comprises atleast one hydroxyl protecting group.
 7. The compound of claim 6, whereinthe at least one hydroxyl protecting group is selected from the groupconsisting of alkyl silyl groups, alkyl ethers, and esters.
 8. Acompound comprising a 4-cyclopentyenyl-2-ethynylthiazole skeleton and aterminal alkyne at the 2-position of a thiazole ring in the4-cyclopentyenyl-2-ethynylthiazole skeleton.
 9. A method of making aCETZOLE compound comprising: subjecting a dibromothiazole to Sonogashiracoupling with TMS-acetylene to obtain a silylated bromothiazole alkyne;subjecting the silylated bromothiazole alkyne to Stille coupling with aniodoalkene to obtain a silylated CETZOLE compound; and desilylating thesilylated CETZOLE compound to obtain a CETZOLE compound.
 10. The methodof claim 9, wherein the dibromothiazole consists essentially of2,4-dibromothiazole.
 11. The method of claim 9, wherein the silylatedbromothiazole alkyne consists essentially of2-((trimethylsilyl)ethynyl)-4-bromothiazole.
 12. The method of claim 9,wherein the iodoalkene consists essentially of3-iodo-2-methylcyclopent-2-enone.
 13. The method of claim 9, wherein thedesilylating comprises mixing a solution of K₂CO₃ and the silylatedCETZOLE compound in methanol for a period of time.
 14. The method ofclaim 9, further comprising the step of subjecting the CETZOLE compoundto stereoselective CBS reduction to obtain a second CETZOLE compound.15. The method of claim 14, wherein the stereoselective CBS reductioncomprises adding borane-Me₂S to a solution ofR-2-methyl-CBS-oxazaborolidine and the CETZOLE compound.
 16. The methodof claim 9, further comprising the steps of: treating the CETZOLEcompound with lithium diisopropylamide and benzyl bromide to obtain asilylated benzyl CETZOLE derivative; and desilylating the silylatedbenzyl CETZOLE derivative to obtain a benzylated CETZOLE compound. 17.The method of claim 16, wherein the silylated benzyl CETZOLE derivativeconsists essentially of5-benzyl-2-methyl-3-(2-((trimethylsilyl)ethynyl)thiazol-4-yl)cyclopent-2-enone.18. The method of claim 16, wherein the desilylating comprises mixingK₂CO₃ and MeOH with the silylated benzyl CETZOLE derivative.
 19. Themethod of claim 16, wherein the benzylated CETZOLE compound consistsessentially of5-benzyl-3-(2-ethynylthiazol-4-yl)-2-methylcyclopent-2-enone. 20.(canceled)
 21. A pharmaceutical composition comprising: a compound ofclaim 1; and a pharmaceutically acceptable carrier, diluent, orexcipient.
 22. A method of treating a mammalian disease comprisingadministering a therapeutically effective amount of a pharmaceuticalcomposition of claim 21 to a subject in need thereof.
 23. The method ofclaim 22, wherein the disease comprises a cancer selected from the groupconsisting of non-small cell lung carcinoma (NSCLC), renal cancer,ovarian cancer, CNS cancer, and melanoma. 24-28. (canceled)
 29. A methodof treating cancer comprising administering to a subject in need thereofan agent that is lethal to cells by a mechanism that does not depend onapoptosis, wherein the agent comprises a compound of claim
 8. 30-32.(canceled)